The ability of microorganisms to produce greater yields of commercially important products at efficient rates can be achieved through metabolic engineering approaches involving amplification, addition or deletion of key enzymes catalyzing relevant metabolic reactions in the concerned metabolic pathways. Redox reactions, catalyzed by enzymes generally referred to as oxidoreductases, in several metabolic pathways are involved in the production of many industrially important compounds. Considering that maintenance of cellular redox balance is the basic requirement for cellular growth and metabolism, as well as for the efficiency with which microorganisms produce commercially important metabolites, manipulation of genes responsible for the maintenance of optimal redox balance provides an additional tool in metabolic engineering of microorganisms. Among various cofactors, pyridine nucleotides namely nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) serve as universal, soluble electron carriers and function as cofactors for several dehydrogenase enzymes. NAD—involved in catabolic pathways and NADP—involved in anabolic pathways undergo reversible reduction concomitant with oxidation (dehydrogenation) of the substrate molecule to function as cofactors for several dehydrogenases. In particular, NAD—in its oxidized (NAD+) and reduced (NADH+H+) forms—functions as the cofactor in over 300 redox reactions and serves not only as an electron acceptor in catabolism but also provides the cell with reducing power in energy conserving redox reactions that occur in aerobic and anaerobic respiration (Foster et al., 1990). A balance in the rates of oxidation and reduction of these nucleotides is a pre-requisite for continuation of catabolism and anabolism since their turnover is higher than their cellular concentrations.
People in the knowledge of prior art are aware of the exergonic pathways which are involved in glucose catabolism under anaerobic conditions results in energy generation—in the form of adenosine triphosphate (ATP)—that is tightly coupled with the generation of NADH+H+ as shown below:Glucose+2NAD++2ADP+2Pi=2Pyruvate+2NADH+H++2ATP+2H2O
Of the two energy conserving reactions of glycolysis, which eventually lead to the formation of ATP, the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphophoglycerate is important since it is concomitant with the reduction of NAD+. Subsequent conversion of pyruvate, a key intermediate in catabolism and the common product of all free energy sources, to either acetaldehyde (by decarboxylation) and further into ethanol or to lactate (by reduction) under anaerobic conditions or to acetyl Co A (by oxidative decarboxylation) under aerobic conditions is largely determined by cellular oxidative status as well as the reactions wherein oxidation of NADH+H+ is achieved to regenerate NAD+. In the absence of regenerating NAD+, the cells would be depleted of electron acceptor required for oxidation of glyceraldehyde 3-phosphate and energy yielding reactions of glycolysis would halt.
In addition to the above role as cofactors, pyridine nucleotide cofactors also regulate gene expression. Thus, limiting NAD synthesis decreased the expression of adhE and increasing NADH concentrations leads to the induction of adhE gene (Leonardo, 1996).
In continuation of the above knowledge accrued from cellular studies, efforts made to engineer the cellular redox potentials by varying relative turnover and yield of NAD+ and NADH+H+ have resulted in significant improvements towards modulating commercial production of metabolites by microorganisms. Most of the currently available strategies of cofactor engineering target (a) the dehydrogenases involved in oxidation and reduction of NAD or (b) enhancing the relative contents of these pyridine nucleotides.
Metabolic engineering approaches made towards amplification or interruption or addition of metabolic pathways has yielded significant results. Importantly, our knowledge of the physiological roles of glycerol in the oxidation of surplus NADH and ethanol in ATP formation has been exquisitely utilized in metabolic engineering.
Notable examples of cofactor engineering by modulating NAD+/NADH ratios towards specific production of targeted metabolites, among others, include:                i) Enhanced availability of NADH+H+ can lead to overexpression of adhE gene (coding for alcohol dehydrogenase) in Escherichia coli, causing enhanced ethanol production under fermentative conditions (Leonardo et al, 1996).        ii) Shift between oxidation or reduction of L-lactaldehyde could be regulated by inhibition of pyruvate dehydrogenase complex by high NADH/NAD ratios (Graef et al., 1999; Baldoma and Aguilar, 1988).        iii) Overexpression of the NADH-dependent glycerol 3-phosphate dehydrogenase (GPD) resulted in a shift of carbon flux towards glycerol production, as well as that of succinate and acetate, at the expense of ethanol production (Remize et al, 1999).        iv) Modulation of glycerol and ethanol yields could also be obtained upon over expressing or disrupting the GPD coding for glycerol 3-phosphate dehydrogenase in Saccharomyces cerevisiae (Nevoigt, 1998).        v) While disrupting GPD 1 resulted in decreased glycerol production and enhanced ethanol formation, overexpression of the gene resulted in significant increase in acetaldehyde formation as well as marked accumulation of pyruvate, acetate, acetoin, 2,3 butanediol and succinate. These alterations could be attained from competitive regeneration of NADH via glycerol 3-phosphate dehydrogenase (Michnik et al, 1998).        vi) Nissen et al (2000) substituted the normal NADPH consuming synthesis of glutamate from ammonium and 2-oxoglutarate in Saccharomyces cerevisiae         vii) with a new pathway marked by consumption of NADH and ATP. The resultant yeast strain produced higher ethanol and lower glycerol yields as compared to the parent wild type strain under anaerobic fermentative conditions.                    Metabolic shift from homolactic to mixed acid fermentation was achieved upon cloning the nox2 gene coding for NADH oxidase from Streptococcus mutans into Lactobacter lactis. Under aerobic conditions, the observed shift in transformants was modulated by the level of NADH oxidation resulting in lowered NADH/NAD+ ratios (Lopez De Felipe et al, 1998).                        
Efforts were also made to regenerate the used cofactors, such as NAD+ and NADH, considering the critical importance of in cofactor-dependent industrial production. These include:                i) In vivo regeneration of NADH employing cellular lysates of Clostridium kluyveri in combination with an aldehyde as an oxidizing agent (U.S. Pat. No. 4,766,071)        ii) Using an electrode to mediate electrochemical regeneration of NADH (U.S. Pat. No. 5,393,615)        iii) Employing coated polymers containing mediators that are covalently linked to the polymeric backbone (U.S. Pat. No. 5,264,092)        
In addition to the above, specific strategies for increasing intracellular production of NADH have also been examined in Escherichia coli. These include (a) feeding carbohydrates with different oxidation states, (b) eliminating pathways using NADH (such as lactate dehydrogenase and alcohol dehydrogenase) that compete for NADH, (c) over expressing NAD+ dependent formate dehydrogenase to regenerate NADH, (d) over expressing nicotinic acid phosphoribosyl transferase that converts exogenously added nicotinic acid to nicotinamide mononucleotide. Of these approaches, varying NADH availability by combining external and genetic means i.e., feeding different carbon sources with different oxidation states to Escherichia coli strains over expressing nicotinic acid phosphoribosyl transferase resulted in enhancing intracellular NADH contents to certain extent. Heterologous expression of Candida boidinii NAD+-dependent formate dehydrogenase in E. coli enhanced the maximal yield of NADH, arising from glucose or sorbitol, from the theoretical maximum of 2 moles to 4 and 4.6 moles per mole of the respective substrate. E. coli strains transformed with heterologous formate dehydrogenase and using sorbitol as the carbon source demonstrated increased ethanol production reflecting on enhanced availability of NADH (Sanchez et al, 2005)
Inventors and scientists in the knowledge of prior art would appreciate that the common motif in the above attempts is to alter NAD/NADH ratios that influence cellular redox balance by targeting one of the several dehydrogenases involved in glycolysis or its related pathways. Thus, manipulation of genes coding for alcohol dehydrogenase or pyruvate dehydrogenase or glycerol 3-phosphate dehydrogenase or formate dehydrogenase was resorted to channel metabolic fluxes towards the formation of microbial metabolites such as ethanol or acetic acid or pyruvate or acetoin or butanediol or succinate. However, all of these dehydrogenases are involved in reducing substrates utilizing reduced NAD (NADH) as the cofactor. Only limited efforts were made to manipulate or over express dehydrogenases involved in the production of NADH.
Glyceraldehyde 3-phosphate dehydrogenase catalyzes the following reaction involving the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate—the first step in the payoff phase of glycolysis.

The acceptor for hydrogen in the reaction is NAD+ involving enzymatic transfer of a hydride ion (:H−) from the aldehyde group of glyceraldehyde 3-phosphate to nicotinamide ring of NAD to yield reduced cofactor NADH; the other hydrogen atom of the substrate appears as H+ in solution. The high amount of standard free energy of the reaction (ΔG0′=−49.3 KJ/mol) is utilized for the formation of ATP in the subsequent reaction wherein 1,3-bisphophoglycerate is converted to 3-phosphoglycerate. Glyceraldehyde 3-phosphate dehydrogenase is the only source of NADH in glycolysis, which is the primary catabolic pathway in the utilization of fermentable sugars and involved in glycolysis and gluconeogenesis by microorganisms. Glyceraldehyde 3-phosphate dehydrogenase is a tetramer distributed in the cytoplasm and cell walls of fermentative producers such as Saccharomyces. In Saccharomyces three unlinked genes (˜1 kbp) entitled TDH1, TDH2, and TDH3 encode related but not identical, catalytically active homoteramers with different specific activities involved in metabolic and structural remodeling (Roberts et al. 2006). While TDH3 protein accounts for 50-60% of total activity, proteins coded by TDH1 and TDH2 respectively account for 10-15% and 25-30% of the total activity.
Among the genes coding for TDH enzymes, TDH1 and TDH2 are located on chromosome X and TDH3 is located on chromosome VII. None of these structural genes is individually essential for cell viability even though the presence of a functional TDH2 or TDH3 gene is mandatory for cell viability. However, it is reported that TDH1 interacts with GTS1 involved in ultradian oscillations of glycolysis and improves anaerobic growth of yeast cultures devoid of glycerol phosphate dehydrogenase (GPD2) gene (Valadi et al. 2004). Despite such extensive information available on the physiological and genetic role of TDH genes, the innate ability of TDH genes towards cofactor engineering has not been exploited towards commercial metabolite production by producer microorganisms. A need therefore still exists for the generation of stable genomic inserts or gene cassettes that encode the necessary enzymes for sugar catabolism for high product formation close to maximum theoretical yield.
Treading on a new line of thought, we made efforts to combine the events related to production and utilization of NADH in order to engineer cofactor requirements related to microbial metabolite production. These experiments resulted in the present invention that describes novel DNA constructs and method for creating a physiological situation wherein greater reduction potential is continuously produced and utilized in a cyclic manner upon over expression of two dehydrogenases involved in production and utilization of NADH. Such an approach would increase specific productivity of industrially important products from sugars by industrial microorganisms. The novelty of the present innovation is also related to the fact that we have engineered two different dehydrogenase enzymes, wherein one is involved in the production of NADH and the other one is involved in the utilization of NADH. This approach is distinctly different from the above-cited approaches wherein only one dehydrogenase that either produces or utilizes NADH has been targeted and used to enhance microbial metabolite production. More importantly, such an approach results in cyclic production and utilization of NADH to augment microbial metabolite production. Because both the enzymes are components of a single metabolic pathway viz., glycolysis, and since both the dehydrogenases are integral components contributing to product formation, such an approach would enhance the channeling of substrate towards product formation.